DESCRIPTION: (Scanned from the applicant's abstract) Growth hormone (GH) is a critical regulator of linear growth and metabolic homeostatis that is selectively synthesized in anterior pituitary somatotropes. The transcription of the GH gene is tightly regulated through the cooperative interactions of a limited repertoire of transcriptional regulators that include Pit-1, C/EBPalpha and the co-activator CBP. The broad objective of the studies outlined in this application is to define when and where the interactions between these proteins occur in the pituitary somatotropes. Much work concerning C/EBPa has focused on its role during adipocyte terminal differentiation; however, little is known about its function in the regulating pituitary cell differentiation. We will use the immortalized murine pituitary cell line GHFT1-5 to establish the importance of the various domains of C/EBPa in its ability to activate the GH promoter as well as control its interactions with Pit-1 and CBP. This culture model will allow the analysis of protein interactions at several levels. The first aim will establish the role of C/EBPa protein domains in directing interactions with other proteins and the GH promoter, as well as examine the effects of PKA and PKC signaling on these interactions. The second aim will determine how the protein domains of C/EBPa affect its subnuclear distribution in living cells and subsequent interactions with Pit-1 and CBP. This will be accomplished by use of proteins tagged with color variants of jellyfish green fluorescent protein. Additionally, I will examine if PKA and PKC signaling affects these interactions in the living cell. These studies will define the key protein domains of C/EBPa that direct interactions with Pit-1 and CBP and yield essential information about the molecular mechanisms that control GH gene expression. It is expected that these studies will have far-reaching implications regarding how transcription factors may influence nuclear organization.